The Origins of Multicellularity and the Early History of the Genetic Toolkit

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ANNUAL The Origins of REVIEWS Further Click here for quick links to Annual Reviews content online, Multicellularity and the Early including: • Other articles in this volume • Top cited articles History of the Genetic Toolkit • Top downloaded articles • Our comprehensive search For Animal Development

Antonis Rokas

Vanderbilt University, Department of Biological Sciences, Nashville, Tennessee 37235; email: [email protected]

Annu. Rev. Genet. 2008. 42:235–51 Key Words The Annual Review of Genetics is online at cell adhesion, cell-cell signaling, transcriptional regulation, animal genet.annualreviews.org phylogeny, choanoﬂagellate, repeated evolution This article’s doi: 10.1146/annurev.genet.42.110807.091513 Abstract Copyright c 2008 by Annual Reviews. Multicellularity appeared early and repeatedly in life’shistory; its instan- All rights reserved tiations presumably required the conﬂuence of environmental, ecolog- 0066-4197/08/1201-0235$20.00 ical, and genetic factors. Comparisons of several independently evolved pairs of multicellular and unicellular relatives indicate that transitions to multicellularity are typically associated with increases in the numbers of genes involved in cell differentiation, cell-cell communication, and Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org

Access provided by Houston Baptist University on 02/03/20. For personal use only. adhesion. Further examination of the DNA record suggests that these increases in gene complexity are the product of evolutionary innova- tion, tinkering, and expansion of genetic material. Arguably, the most decisive multicellular transition was the emergence of animals. Decades of developmental work have demarcated the genetic toolkit for animal multicellularity, a select set of a few hundred genes from a few dozen gene families involved in adhesion, communication, and differentiation. Examination of the DNA records of the earliest-branching animal phyla and their closest protist relatives has begun to shed light on the origins and assembly of this toolkit. Emerging data favor a model of gradual assembly, with components originating and diversifying at different time points prior to or shortly after the origin of animals.

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INTRODUCTION specific genes (83), and the elaborate coordi- nation of developmental processes, made them From the simple, undifferentiated bacterial fil- stand out as one of the most complex inventions Complexity: a aments to the macroscopic multicellular forms of multicellularity (7, 19, 46, 70). Elucidating problematic term used seen in animals, plants, and fungi, the 25 or the enigma of the origins of multicellularity in in a variety of different so instantiations of multicellularity extant to- contexts; here it is used animals requires, to a large extent, solving the day exhibit a remarkable diversity in genotypic to simply denote enigma of the origins of their development. and phenotypic complexity (5, 51) (Table 1). increases in numbers But what is the genetic basis of animal multi- of cell types, body size, For example, the multicellular forms observed cellularity and development? Animal genomes life-cycle stages, genes, in prokaryotes are architecturally and morpho- contain thousands of genes involved in carrying or protein domains logically relatively simple, characterized by the out vital routine tasks, such as metabolism and presence, at their most elaborate manifesta- cell division. Many of these genes are shared tions, of a few distinct cell types (9). Similar lev- across eukaryotes and predate the origin of ani- els of complexity are observed in most cases of mals per se (23, 60), but some underwent exten- eukaryotic multicellularity (7, 9, 103). The in- sive gene duplications and evolved new roles in dependent transitions to multicellularity from the construction and patterning of animal bod- unrelated unicellular ancestors offer a unique ies. These genes comprise the genetic toolkit opportunity for comparative study, especially at for animal development (20, 57), a select set the molecular level. We start by identifying the of a few hundred genes from a few dozen gene general conditions favoring the emergence of families involved in three key processes: cell dif- multicellularity, its origins, and its signature in ferentiation, cell-cell communication, and cell the DNA record. adhesion. Examples of toolkit components in- Most multicellular lineages are charac- clude the Hox transcription factors (35), the cell terized by relatively simple architectures and signaling families of Wnts and receptor tyrosine morphologies. However, on a few separate kinases (53, 62), as well as the gene families of occasions, the transition to multicellularity has cadherins and integrins, which are involved in burgeoned into macroscopic, architecturally cell adhesion (1, 72). Understanding the origins complex body plans (e.g., plants, fungi, and ani- and assembly of the genetic toolkit required for mals) (9). In animals, for example, the evolution animal multicellularity and development is the of several differentiated cell types generated by second and central focus of this review. the specific expression of a number of cell-type–

Table 1 The genetic and phenotypic complexity of select, independently evolved, multicellular bacterial and eukaryotic lineages Representative Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org 1 Access provided by Houston Baptist University on 02/03/20. For personal use only. Lineage Cell type number species Gene number Genome size (Mb) References Actinobacteria 3 Streptomyces coelicolor 7825 9 (8) Cyanobacteria 3 Nostoc punctiforme 7432 9 (69) Myxobacteria 3 Myxococcus xanthus 7388 9 (14, 38) Cellular slime molds 3 Dictyostelium discoideum 13,541 34 (7, 32) Animals 3–122 Drosophila melanogaster 13,733 200 (7, 24) Fungi 3–9 Coprinus cinereus2 13,544 37.5 (7) Volvocine green algae 2 Volvox carteri3 15,544 140 (7) Plants 5–44 Arabidopsis thaliana 25,498 125 (24, 100)

1The ﬁrst three lineages are bacterial; the rest eukaryotic. 2Genome unpublished; data retrieved from the Broad Institute (http://www.broad.mit.edu/). 3Genome unpublished; data retrieved from the Joint Genome Institute (http://www.jgi.doe.gov/).

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Insights from paleontology, ecology, and multicellular setting by functional specializa- phylogenetics provide the temporal, environ- tion, at least in principle. mental, and historical context within which we In several instances, theoretical expectations Myxobacteria: a can understand the emergence of multicellu- have been put to the test. The results have group of multicellular larity. Similarly, dramatic advances in develop- demonstrated that several reasons typically as- δ-proteobacteria, also mental genetics and comparative genomics are sociated with transitions to multicellularity, known as significantly enriching our understanding of the such as predation avoidance or higher feeding myxomycetes, with a genetic changes associated with multicellular efficiency, do indeed confer a selective advan- complex life-cycle during which they transitions, and of the origins of the animal de- tage over unicellularity. For example, a number construct a velopmental program in particular. The body of algal species were able to evolve multicellu- multicellular fruiting of facts now emerging has shed ample light on larity when grown in culture in the presence body the tempo and pattern of this pivotal period in of predators, thus dramatically reducing their life’s history and is setting up the framework chances of being eaten (11, 47, 66). Similarly, within which we can understand the origins and Volvox algae (61) and myxobacteria (88) have assembly of the genetic toolkit for animal mul- been shown to be at advantage when multicel- ticellularity and development. lular because of their ability to better utilize available nutrients. Most manifestations of multicellularity are THE EVOLUTION OF relatively simple in architecture, involving MULTICELLULARITY: A only a very small number of cell types (19, 58) COMPARATIVE PERSPECTIVE (Table 1). Cell-type determination typically occurs via the action of a small number of regu- Why Did Multicellularity Evolve? latory proteins (49). However, the large number It is statistically unlikely that complex pheno- of regulatory proteins present in both prokary- types arise repeatedly by chance (25). Thus, otes and eukaryotes suggests that, from a ge- from a comparative perspective, the multiple nomic point of view, these organisms have the origins of multicellularity in a wide variety of potential to generate a much larger number of organisms from distinct evolutionary lineages cell types than those actually observed (19). So underscore the notion that key aspects of this why do most multicellular organisms possess so phenotype are likely to be, under certain con- few cell types? Although it is difficult to address ditions, selectively advantageous. Considerable this question a posteriori, a plausible explana- attention has been devoted to identifying what tion may be that there was no selective pressure these aspects or conditions may have been, with for early microscopic multicellular organisms a variety of factors implicated as plausible con- to further increase their size, and consequently tributors to multicellularity’s repeated inven- diversify their pool of cell types beyond a small Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org

Access provided by Houston Baptist University on 02/03/20. For personal use only. tion (39, 51). Theoretical work suggests that a number (39). Support for this explanation multicellular existence could have been advan- comes from both theory and empirical obser- tageous by reducing predation (97), improving vations, which indicate that differentiated cell the efficiency of food consumption (9), facil- types are generally more likely to evolve in itating more effective means of dispersal (9), larger multicellular organisms (7, 10, 94, 105). limiting interactions with noncooperative indi- Any multicellular organism increasing its viduals (71, 77, 78), or dividing labor (71). For size is likely to encounter a trade-off between example, unicellular lifestyle conflicts, such as the conflicting selective pressures from escap- the dependence of flagellum-induced motility ing predation and avoiding the consumption and mitosis on the same molecular machinery of the additional energy required to maintain (16, 51), or the requirement for spatial or tem- a larger body size. This conflict has been beau- poral separation of certain metabolic processes tifully illustrated by a laboratory experiment (39, 45), could have been easily resolved in a where, in the presence of a predator, a culture

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of unicellular algae evolved multicellularity in three distinct cell types (17). Dictyostelid cel- fewer than 100 generations (11). During the lular slime molds are thought to have diverged course of the experiment, the number of cells prior to the splitting of fungi and animals (98), Cyanobacteria: a group of per multicellular organism varied between 4 to but exactly when multicellularity arose in this photosynthetic more than 100, with the population eventu- lineage is unknown. In contrast, the Volvocine bacteria that contains ally stabilizing to 8-celled bodies despite be- green algae, which represent one of the most unicellular, ing much higher in earlier generations. Impor- recent inventions of multicellularity, diverged undifferentiated tantly, an 8-celled body is just big enough to from their unicellular relatives a mere 0.05 bya multicellular (filamentous), and confer escape from predation (11). (56). Molecular clock estimates place the ori- differentiated gin of the complex multicellularity observed in multicellular species plants, animals, and fungi some time between When Did Multicellularity Evolve? bya: billion years ago 1.0–0.4 bya (30), with unambiguous fossils from Actinobacteria: a Judging from these potential advantages of a each of these lineages appearing between 0.6– group of high G+C multicellular lifestyle over a unicellular one, 0.4 bya (50, 102, 109). gram-positive, mostly multicellularity would be expected to appear multicellular bacteria, relatively early in the course of life’s evolu- Evolution of complex multicellular lineages: also known as tion. Evidence from the fossil record seems too few, too late. Examination of both the actinomycetes, that is frequently found in to corroborate this expectation. Simple fila- bacterial and eukaryotic fossil record strongly soils mentous manifestations of multicellularity are indicates that the first experiments in multi- Proterozoic: an era found in the early fossil records of both bacterial cellularity were already present much earlier in the geologic time (104) and eukaryotic lineages (58), although the than the emergence of complex multicellular- scale that spans from more complex instantiations of multicellularity ity (19, 58). Examination of Earth’s history in- about 2.5 bya to the in both lineages appeared much later. dicates two major events immediately prior to beginning of the On the bacterial stem of the tree of life, fil- the origin of complex multicellularity, namely Cambrian period (at 0.54 bya) and during amentous cyanobacteria with distinct cell types predation (82, 97) and a sharp increase in oxy- which eukaryotes first first appeared approximately 2.5–2.1 billion gen levels (42), that may have contributed to appear in the fossil years ago (bya) (101); their earliest examples its relatively late appearance. For example, the record were fossilized resting cells that can withstand abundance of oxygen in Earth’s shallow oceans Protist: a generic environmental stress, also known as akinetes, was an order of magnitude lower than current name used to describe from the genus Archaeoellipsoides (4, 101). The levels until approximately 0.85 bya (42), and any microscopic fossil record is silent for the other two groups would thus have imposed severe constraints on eukaryotic organism of multicellular bacteria, actinobacteria and the evolution of macroscopic bodies with high Green algae: a large myxobacteria, but estimates based on the 16S energy demands. Similarly, multiple lines of ev- and diverse group of unicellular and ribosomal DNA molecular clock offer approx- idence argue that it may have been only after the multicellular of imate dates of origin. Actinobacteria appear to emergence of predators that the selective ben- Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org

Access provided by Houston Baptist University on 02/03/20. For personal use only. photosynthetic be almost as old as differentiated multicellular efit of a larger size would have been sufficient organisms cyanobacteria, with an estimated date of origin to drive the evolution of complex multicellular approximately 2.0–1.9 bya (33), whereas mul- forms (11, 47, 66, 97). Examination of the fos- ticellular myxobacteria appeared much later in sil record suggests that the first predatory eu- the Proterozoic, close to 1.0–0.9 bya (95). karyotes appeared approximately 0.75 bya (82, On the eukaryotic stem, filamentous protists 97), a date strikingly contemporaneous with the first appear in the fossil record very soon af- emergence of the first ancestors of fungi and ter the appearance of the first unicellular eu- animals (30, 82). karyotes some 1.8 to 1.2 bya, and differentiated multicellular protists appeared no later than 1.2 bya (58). An example of the multicel- How Did Multicellularity Evolve? lular complexity exhibited by these early fossils Given that multicellularity has evolved repeat- is Bangiomorpha, a red algal fossil with at least edly from independent unicellular lineages,

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comparisons of the gene sets of multicellular have dramatically diversified in numbers and and unicellular pairs allow us to infer the likely probably also in function in multicellular lin- gene set of the unicellular ancestor as well as the eages. Third, some of the components found Protein domain: changes that have taken place during the evolu- in abundance in multicellular lineages are ab- polypeptide chains tion of the multicellular species. Although the sent from their unicellular relatives and likely that exhibit structural, comparative approach is very powerful, infer- represent novel innovations. functional and ence of molecular events that have transpired A number of studies have examined the in- evolutionary unity; over hundreds of millions of years can be chal- dependent transitions to multicellularity in the they are the building block(s) of proteins lenging. This is likely to be the case if ei- bacterial lineage (8, 38, 69). Comparisons of ther of the lineages compared have diverged differentiated multicellular cyanobacteria (e.g., so long ago that accurate identification of an- Nostoc and Anabaena) with their undifferen- cestral states or direction of change is difficult tiated multicellular (e.g., Trichodesmium) and (74), or if their genomes have become stream- unicellular (e.g., Synechocystis, Synechococcus, and lined as a consequence of adaption to special- Prochlorococcus) relatives revealed large increases ized lifestyles (29). Finally, note that not all in- in the genes involved in signal transduction stantiations of multicellularity are the same, and and transcriptional regulation (45, 69, 107). For that they do differ in important details. For ex- example, whereas the number of transcription ample, multicellularity in Volvocinegreen algae factors in differentiated multicellular species likely evolved as a consequence of incomplete ranged between 124 and 172, their number separation after cell division, whereas in Dic- in undifferentiated multicellular or unicellular tyostelid cellular slime molds multicellularity species ranged between 18 and 64 (107). Evi- evolved as a consequence of aggregation (104). dence for participation of these additional genes Thus, any expectation that gene families par- in the manifestation of multicellularity comes ticipating in cell adhesion in the two lineages from analysis of levels of gene expression, which would show similar trends relative to their uni- shows that they are up-regulated during the cellular relatives simply because both are mul- differentiation process (18). A similar trend of ticellular would likely be unfounded. an increase in cell-cell signaling and transcrip- These caveats notwithstanding, several tional regulation genes is seen in comparisons studies have compared the DNA records of uni- of the multicellular myxobacterium Myxococ- cellular and multicellular species (8, 38, 45). cus xanthus with its unicellular δ-proteobacterial These first comparisons have investigated a relatives (38). A dramatic increase in regulatory wide variety of characteristics thought to be genes is also seen in comparisons of the mul- correlated with transitions to multicellularity, ticellular actinobacterium Streptomyces coelicolor such as the presence of protein domains in- with its unicellular relatives, where the num- volved in characteristic multicellular functions ber of σ transcription factors, for example, is Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org

Access provided by Houston Baptist University on 02/03/20. For personal use only. (e.g., cell-cell signaling and communication, approximately ﬁvefold higher (8). cell-cell adhesion, and transcriptional regula- A large fraction of the additional genes tion) or an increase in their gene family com- associated with cell-cell signaling and tran- plexity. Data from these comparisons provide scriptional regulation observed in these three key insights to understanding the origins multicellular-unicellular comparisons can be and assembly of the genetic toolkits associated accounted for by gene duplication (8, 38). with transitions to multicellularity. First, many, For example, genomic analysis of M. xanthus but not all, of the molecular components of identiﬁed more than 1500 duplications that the genetic toolkit are also present in the DNA occurred during the transition to multicellu- records of unicellular relatives, which suggests larity, and determined that cell-cell signaling that these components were likely present in and regulatory genes underwent 3 to 4 times their last common (unicellular) ancestor. Sec- as many duplications as would be expected ond, several of these preexisting components by chance (38). Although the origins of

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many of these genes predate multicellularity, through sequence duplications. This topic has their function in the unicellular relatives is been examined in great depth (20, 27) and is not not always obvious. Take, for example, the discussed further here. The second implication, gene cluster identified in the differentiated pertinent to the scope of this review, is that if multicellular cyanobacterium Anabaena to we wish to retrace the early evolution of the ge- regulate differentiation and pattern formation netic toolkit, examination of the DNA records of heterocysts (110). The cluster is conserved of bilaterians alone is not likely to suffice. We across both differentiated and undifferentiated have to look deeper into life’s evolutionary ge- multicellular cyanobacteria, but absent from nealogy to seek its origins in the DNA records unicellular ones, suggesting that its ancestral of the morphologically simplest animal phyla role (likely still present in undifferentiated (such as poriferans, ctenophores, placozoans, filamentous species) was a more general one in and cnidarians), or even further back in time, in filamentation (110). The study of gene families the closest protist relatives of animals (such as with key roles in multicellularity in unicellular choanoflagellates and ichthyosporeans). To do relatives will be critical for understanding the so requires that we first reconstruct the origin genes’ ancestral functions and their cooption and evolutionary diversification of major animal to the multicellular developmental program. groups, with special emphasis on resolving relationships among early-branching lineages and on identifying the protist relatives of animals. ORIGINS AND EVOLUTION OF Examination of the fossil record reveals a THE GENETIC TOOLKIT FOR Precambrian origin of sponge, cnidarian, and ANIMAL MULTICELLULARITY bilaterian body fossils, whereas the first fossil AND DEVELOPMENT occurrences of the uniquely distinct bilaterian Important clues to the origins and assembly body plans of phyla such as arthropods, chor- of the genetic toolkit may be gleaned through dates, mollusks, echinoderms, and annelids are careful comparisons of the DNA records of found in Cambrian-age rock strata (102). While extant animal phyla and their closest, mostly fossils are our only direct window to the past, unicellular, protist relatives. Notwithstanding their utility in reconstructing the evolutionary a major expansion of the genetic toolkit dur- diversification of animals may be limited. For ing early chordate evolution (43), examination example, the fossil record is silent regarding of the DNA record of protostomes (such as ne- the earliest appearances of unicellular and colo- matodes, fruitflies, and mollusks) and deuteros- nial relatives of animals (58). Perhaps more im- tomes (such as echinoderms, tunicates, and portantly, fossils can only impose lower bounds vertebrates) shows that the genomes of bilat- on divergence estimates because recognizable erally symmetrical animals are characterized by body fossils always appear after the cladoge- Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org

Access provided by Houston Baptist University on 02/03/20. For personal use only. fairly similar toolkit gene sets (57, 73). Thus, netic events that give rise to distinct lineages the toolkit’s essential components were proba- (57), whereas the time interval between these bly already in place by the origin of bilaterian two events is unknown (15). Thus, reconstruct- animals near the end of the Proterozoic (59). ing the evolutionary diversification of animals The presence of the genetic toolkit in the bi- and their relatives requires that we turn our at- laterian ancestor has two serious implications. tention to the DNA record of extant represen- The first is that the bewildering diversity of bi- tatives of these lineages. laterian body plans was generated by further The DNA record has proven exceptionally tinkering of the basic genetic toolkit, especially useful for charting the tempo and pattern of via the modification of patterns of gene expres- life’s evolutionary history, and has helped to sion through the evolution of cis-regulatory el- clarify the tempo and mode of an enormous ements, as well as via the acquisition and sub- number of key evolutionary events (26). Con- sequent functional diversification of new genes trary to the progress observed in the resolution

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a Bilaterians b Placozoans c Bilaterians Ctenophores Demosponges Demosponges Cnidarians Placozoans Cnidarians Cnidarians Demosponges Calcareous poriferans Ctenophores Bilaterians Hexactinellid poriferans

d Bilaterians e Bilaterians f Animals Hexactinellid poriferans Hexactinellid poriferans Ministeria Cnidarians Calcarean poriferans Demosponges Choanoflagellates Placozoans Demosponges Ichthyosporeans Choanoflagellates Cnidarians Ichthyosporeans Corrallochytreans

g Animals h Animals

Choanoflagellates Choanoflagellates

Capsaspora Capsaspora

Ichthyosporeans Ichthyosporeans

Figure 1 A representative sample of alternative phylogenetic scenarios among early-branching animals and the closest protist relatives of animals. Phylogenies from (a) (31), (b) (28), (c) (79), (d ) (86), (e) (40), ( f ) (98), (h) mitochondrial genome phylogeny from (90), and ( g) nuclear gene phylogeny from (90).

of innumerable other branches of the tree imal tree, a placement in agreement with ob- of life, the early history of animal diversiﬁ- servations that poriferans are the ﬁrst animals cation has proven recalcitrant to resolution to appear in the fossil record (13, 37, 75). Fur- (Figure 1). Below, we review the state of knowl- ther support for this placement comes from the edge in two parts of the animal tree that are crit- remarkable cytological similarities shared be-

Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org ical to this review, namely the early branching tween choanocytes, the feeding cells of sponges, Access provided by Houston Baptist University on 02/03/20. For personal use only. animal lineages, and the closest protist relatives and a phylum of unicellular and colonial pro- of animals. tists known as the choanoflagellates (48, 64) (see below). In contrast, other molecular studies point to a clade of early-branching animals The Diversification of that group with bilaterians. In these studies, Tri- Early-Branching Animals choplax adherens, the single representative of the Most attempts to reconstruct early animal his- enigmatic phylum of placozoans, features as the tory raise intriguing questions about the evo- earliest branching phylum on the sister clade to lution of animal development (Figure 1a–e). bilaterians (28). Placozoans exhibit a very sim- For example, several molecular (6, 12) and mor- ple body plan characterized by just four cell phological (13) studies have identified porifer- types, an absence of organs, and axis of sym- ans as the earliest-diverging branch of the an- metry (7, 28). Other more complex scenarios

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have also been proposed that include, for ex- animals were compressed in time (73, 86), more ample, poriferan paraphyly (12, 75), cnidari- than 600 million years later it might matter little ans as the sister group to bilaterians (75), or to know the exact relationships between most ctenophores as the bilaterian sister group (76). phyla to understand the evolution of the molec- More radical placements have also been put for- ular tool kit that enabled the evolution of the ward. For example, a recent analysis of exten- body plans of the 35 or so animal phyla. sive molecular data identified ctenophores as the earliest branching phylum of the animal tree (31). Given that ctenophores are morphologi- The Search for the Protist cally and developmentally much more complex Relatives of Animals than either poriferans or placozoans (67), their Which are the closest extant relatives of an- placement would require either loss of this com- imals? Several studies have pointed to five plexity in the placozoan and poriferan lineages eukaryotic lineages: the Ministeria clade, the or its independent gain in ctenophores (31). Capsaspora clade, the corallochytreans, the How we reconcile these sharply contrast- choanoflagellates, and the ichthyosporeans ing views of early animal history remains an (also known as mesomycetozoans) (89, 90, 98). open question. The lack of inclusion of rep- Although a consensus view of their evolution- resentative taxa from key lineages frequently ary affiliations and placement with respect to makes comparisons between studies problem- animals has yet to emerge, these studies have atic. For example, neither Trichoplax nor repre- evinced that all these lineages have deep ori- sentatives of two of the three poriferan classes gins (89, 90, 98). These five protist lineages (31) were included in the study that identified exhibit a wide variety of lifestyles: Capsas- ctenophores as the earliest branching lineage pora and ichthyosporeans are parasitic, whereas (Figure 1). Another puzzling feature of several choanoflagellates, corallochytreans, and Minis- of these studies is that their (contradictory) con- teria are all free-living (68, 98). Differences are clusions are strongly supported. Unfortunately, also observed in their morphological charac- concatenations of large gene numbers will al- teristics: Corallochytreans and Ministeria lack most always yield high clade support values, flagellae, but choanoflagellates are flagellated even if the underlying support for one topology (68, 98). over another is marginally better (84). Thus, In the absence of precise phylogenetic high clade support values do not always guar- knowledge, identifying which of these protist antee that the topology obtained is correct (80, lineages may offer the best comparison with 84, 87, 99). The list of studies reporting abso- animals requires further examination of their lute support for alternative conflicting animal biology and lifestyle. The study of Ministeria phylogenies has grown in recent years, a result and corallochytreans presents practical chal- Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org

Access provided by Houston Baptist University on 02/03/20. For personal use only. most likely attributable to the increased data. lenges because both groups are difficult to cul- On the basis of experimental and simula- ture, especially in bacteria-free environments tion analyses, we have proposed that early an- (89). In contrast, comparisons of the multi- imal evolution was likely an evolutionary radi- cellular and unicellular lifestyle based on the ation (86). This result is compatible with the genetic makeup of ichthyosporeans and Cap- fossil record (102), and can explain the con- saspora present analytical challenges, as their flicting conclusions reached by other studies as DNA records are likely to have been influenced short-stemmed, long-branched phylogenies are by the parasitic lifestyles of these organisms notoriously difficult to resolve (34). The impli- (68). Nevertheless, under certain conditions, cations of a radiation during early animal evolu- ichthyosporeans form multicellular structures tion for understanding the origins and assembly (89), suggesting that their genomes may indeed of the toolkit of animal development and mul- offer vital clues to the molecular origins of multicellularity are profound (84). If the origin of ticellularity.

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A number of attributes indicate that the most ample, a comparison of Caenorhabditis elegans valuable lineage for comparative purposes may with S. cerevisiae revealed the presence of several be the phylum of choanoflagellates. Cell struc- novel domains involved in transcriptional reg- ture in choanoflagellates, a bulbous cell body ulation and extracellular adhesion in the worm surrounded by a protoplasmic, apical collar that proteome, as well as an enrichment in domains encircles their single flagellum, is thought to shared by both organisms (22). In agreement be ultrastructurally remarkably similar to that with inferences from studies on bacterial tran- of choanocytes, the feeding cells of sponges sitions to multicellularity, the transition to mul- (48, 64). This similarity has given rise to multicellularity in animals may not have required tiple suggestions that their cellular morphol- the evolution of new genes but rather an in- ogy may be reminiscent of that of the unicel- crease of complexity of certain gene families, lular ancestor of animals (65, 93). Importantly, either through the evolution of novel domains several recent phylogenetic studies have eluci- or the further shuffling of the domain set al- dated the relationships between poriferans and ready available. choanoflagellates. Several lines of evidence in- We propose three models to explain the dicate that choanoflagellates are very close rel- origin and assembly of the animal genetic atives of animals, counter to the hypothesis that toolkit, preanimal, pan-animal, and within- they may be a lineage secondarily derived and animal (Figure 2). According to the preanimal simplified from poriferan ancestors (51, 55, 85, model, the origins of the toolkit predate the ori- 86). gin of animals with some, if not all, components All 125 choanoflagellate species known to of the toolkit present in protist relatives of an- date have retained a free-living lifestyle, and imals. The pan-animal model argues for an ex- representatives of each of the three families in plosive origin of the toolkit; the toolkit is absent the phylum exhibit considerable phenotypic di- in the close relatives of animals but all compo- versity, mostly associated with external cell or- nents are present in even the earliest-branching namentations and covers (52). Importantly, a animals. Finally, the within-animal model sug- number of choanoflagellate species form multi- gests that the genetic toolkit was incrementally cellular (colonial) structures. An interesting ex- assembled during early animal evolution, with ample is offered by Proterospongia, a choanoflag- some, but not all, components of the toolkit ellate with a two-phase life cycle, of which one present in early-branching animals. is multicellular, and with a total of four distinct Data emerging from several studies strongly cell morphologies (65). The multicellular stage indicate that different components of the ge- has the shape of a gelatinous mass, with col- netic toolkit originated and diversified at differ- lared cells on the surface and collarless ones at ent time points during the transition to animal its interior (44). multicellularity (1, 51, 53, 54, 63, 72), suggest- Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org

Access provided by Houston Baptist University on 02/03/20. For personal use only. ing that more than one of the proposed models may be required to explain its origins and as- The Origins and Assembly sembly (Figure 3). This inference was recently of the Genetic Toolkit validated by the genome sequencing and analy- An early genomic comparison of the uni- sis of the unicellular choanoflagellate Monosiga cellular yeasts Saccharomyces cerevisiae and brevicollis (55). Comparisons of the choanoflag- Schizosaccharomyces pombe with humans, flies, ellate genome with animal and fungal genomes and worms found only three highly-conserved suggest that most cell-adhesion gene families genes present in animals that did not have ho- clearly predate animal origins, thus conform- mologs in unicellular yeasts (106). However, ing to a preanimal model, whereas most cell- when protein domains rather than genes are cell signaling and differentiation gene families used as the units of comparison, large-scale dif- postdate animal origins, which supports either ferences in content become apparent. For ex- a within-animal or a pan-animal model (62, 72).

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a “Pre-animal” model b “Within-animal” model c “Pan-animal” model

Bilaterian animal (e.g., chordate, arthropod, annelid) Protist relative (e.g., choanoflagellate, ichthyosporean) Early-branching animal (e.g., poriferan, cnidarian, placozoan, ctenophore)

Figure 2 Three alternative models for the evolution of gene family complexity of the genetic toolkit for animal multicellularity and development. (a) The pre-animal model, (b) the within-animal model, and (c) the pan-animal model. The different colors represent different members of the same gene family, whereas the different shapes correspond to the different clades in which protein members are found (e.g., bilaterians, early-branching animals, protists). For example, in the pre-animal model four proteins of the same protein family are present in both bilaterian (circles) and early-branching animals (squares), but only one member of the protein family—the most basal—is present in eukaryote relatives (star).

For example, whereas the cell adhesion family animal cells. Examination of the choanoflagel- of cadherins is very diverse in choanoflagellates late proteome suggests that the gene machinery (1), and thus likely to have been similarly so participating in adhesion in animals was likely in the unicellular common ancestor shared by well developed in the unicellular ancestor of choanoflagellates and animals, beta integrins or animals and choanoflagellates. Most of the do- Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org

Access provided by Houston Baptist University on 02/03/20. For personal use only. Wnts are entirely absent from choanoflagellates mains typically found in animals are present in (55). choanoflagellates, including those of cadherins, C-type lectins, immunoglobulins, and α integrins (1, 54, 55). However, what is the function The indelible stamp of lowly origin of the of such a diverse set of adhesion molecules in a cell adhesion machinery. The adhesion of unicellular organism that is not known to form animal cells to their neighbors and the extracel- cell-cell connections? Examination of the ex- lular matrix is a fundamental aspect of animal tracellular localization of two choanoflagellate multicellularity. A few major classes of genes cadherins reveals their presence, and colocal- such as the cadherins, the integrins, the selectins ization with actin, at the organism’s apical collar (e.g., C-type lectins), and the immunoglobu- (1). The choanoflagellate collar serves as a food- lin superfamily (e.g., fibronectin type III do- catching device onto which bacteria are latched mains) play a key role in mediating adhesion in and transferred toward the cell (44), raising the

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Bilaterians Early-branching metazoans Deuterostomes Protostomes Vertebrates Cephalochordates/ Arthropods Nematodes CnidariansPlacozoans Poriferans Choanoflagellates Tunicates

Cadherins 127 32 17 46 23

b integrins 11 8 2 2 5 3 0

Wnts 1913 8 5 12 0

Homeobox 290 87 177 118 154 36 31 2

T-box 17 9 8 21 13 0

Figure 3 Different components of the genetic toolkit originated and diversified at different time points during the transition to animal multicellularity. For example, whereas cadherins are as diverse in choanoflagellates as they are in flies, several other gene families are either absent (e.g., T-box) or less diverse (e.g., Homeobox) in choanoflagellates relative to animals. It is not known whether all phyla within the other major clades exhibit similar levels of gene family complexity. Data for cadherins from (1), for Wnt from (62), for poriferan homeobox genes from (63), and for T-box genes from (55, 108). All other numbers were calculated by searching the proteomes of representative species with the corresponding domains as constructed by the PFAM database (36), using an E-value cut-off of 10−5.

possibility that the origins of this major animal of multicellularity in animals, and a handful cell adhesion gene family may lie in molecules of core signaling pathways, such as nuclear originally invented for prey capture (1). hormone receptors, Hedgehog, Wnt, TGFβ, Several genes participating in the formation Notch, and receptor tyrosine kinases, are in- of the extracellular matrix are also well con- volved in its materialization (81). In contrast to served and predate animal origins, including the preanimal origin of most of the gene ma- collagen, laminins, and fibronectins (55). Per- chinery associated with cell adhesion, the ori- haps the most spectacular example of the deep, gins of signaling pathways were an animal inno- preanimal origins of some of these gene fam- vation (55). Several of the pathways (e.g., Wnt ilies is offered by collagens, the most abun- and TGFβ) are absent from choanoflagellates, dant protein family in the mammalian body although they appear to be present in early- (41), homologs of which are found not only branching animals (2, 55). Perhaps surprisingly, in choanoflagellates, but also in the animal sis- Wnts exhibit remarkable gene family complex- ter kingdom, the fungi (21). However, inte- ity in early-branching animals; the cnidarian grins, one of the major receptors of collagen, are Nematostella vectensis contains gene representa- not found in fungi (41). Furthermore, whereas tives for at least 11 of the 12 recognized Wnt

Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org in animals integrins are functional as het- subfamilies (62) (Figure 3). This complexity of Access provided by Houston Baptist University on 02/03/20. For personal use only. erodimers constructed out of α and β subunits Wnts in early-branching animals argues for an (41), the choanoflagellate genome contains only episodic, pan-animal origin of this gene fam- α integrins (55). This finding suggests that the ily, although the sudden increase in complexity interaction between integrins and collagen in may be an artifact of the lack of thorough sam- choanoflagellates may differ from their inter- pling for these genes in placozoans, poriferans, action in animals, and that its study may yield or ctenophores. important insights about the evolution of ani- Nonetheless, distinct domains of certain mal cell adhesion to the extracellular matrix. pathways are discernible in the choanoflagellate genome (e.g., Notch, Hedgehog, and MAPK), The early animal origins of the cell-cell suggesting that animal signaling molecules signaling machinery. Cell communication is may have evolved, at least partially, through critical for the generation and maintenance the shuffling and co-option of pre-existing

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domains. The evolutionary origin of the mals has already yielded important insights into Hedgehog protein offers a telling example of the tempo and mode of the genesis of the ge- the likely importance of this process and its po- netic toolkit and the likely functions of the gene TF: transcription factor tential role in the genesis of the genetic toolkit. machinery that predated but was co-opted for Bilaterian Hedgehog proteins are composed of multicellularity in the time antecedent to the two domains, aptly known as the hedge and the transition. hog (3). Choanoflagellates have only the hog Questions about deep origins and major domain, whereas poriferans and cnidarian pro- evolutionary transitions were once thought to teomes contain both domains but as parts of be, for all practical purposes, imponderable. distinct proteins, suggesting that the Hedge- Important advances in our understanding of hog protein likely first evolved through domain how to read and make sense of Earth’s early life shuffling in an early animal ancestor (3, 55, 96). and environmental history, the theory and experiments associated with transitions in individ- The emergenece of novel transcriptional uality, the genetics of animal development, and regulation machinery in the animal lineage. finally the DNA record of a multitude of crea- Transcriptional regulation is of crucial impor- tures have changed all this. Our understanding tance in the manifestation of animal multi- of the life and weather in Proterozoic oceans cellularity and development (20, 27). Here is is continuously improving, the theoretical and where the protist heritage of the choanoflagel- practical conditions for unicellular to multicel- late proteome is most fully exposed, as its pro- lular transitions are being worked out, at the teome contains the standard set of transcrip- same time as comparisons of several indepen- tions factors (TFs) observed across eukaryotes, dent evolutions of multicellularity are revealing with most of the well-known animal TFs ab- telltale molecular changes in key parts of the sent (1, 55). In contrast, examination of the pro- molecular machinery. teomes of early-branching animals shows an ap- Much, however, remains to be understood. preciable increase in TF family complexity, with If the origins of some of the gene machinery both poriferans and cnidarians containing sev- that makes us multicellular can be found in our eral representatives of the Fox, T-box, Paired, unicellular relatives, how did it get there in the and POU families (63, 108). However, tran- first place and what was its original function? scription factor family complexity among early- How are we to reconcile the conflicting evolu- branching animals is not equal; cnidarians are tionary scenarios of relationships among early- qualitatively (e.g., Hox class homeobox genes branching animals with the genesis and early are present only in cnidarians) and quantita- evolution of the genetic toolkit? Was the ge- tively more complex relative to poriferans and netic toolkit causal in the evolution of animal placozoans (55, 91) (Figure 3). Further exami- multicellularity or simply its product? What Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org

Access provided by Houston Baptist University on 02/03/20. For personal use only. nation of the proteomes of early-branching ani- was the relative contribution of extrinsic (eco- mal phyla is likely to be crucial in understanding logical and environmental) and intrinsic (ge- the origins of animal transcription factors. netic) factors in the origins of animal multicellularity? If what has been achieved so far is any guide for how future work will progress, CONCLUSION the prospects could not be more promising. To In summary, examination of the DNA record of quote the great embryologist Hans Spemann several multicellular lineages has already iden- (92): “What has been achieved is but the first tified several important molecular trends asso- step; we still stand in the presence of riddles, ciated with transitions to multicellularity. On but not without hope of solving them. And rid- the animal front, the comparison of choanoflag- dles with the hope of solution—what more can ellates with early-branching and bilaterian ani- a scientist desire?”

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SUMMARY POINTS 1. Multicellularity has repeatedly evolved, at different times, in several prokaryotic and eukaryotic lineages. 2. Several different ecological, environmental, and genetic factors have likely contributed to the emergence of most multicellular lineages. 3. Examination of the DNA record of several lineages suggests that multicellular transitions are frequently characterized by increases in gene family complexity of molecules involved in one of three key processes for multicellular growth and differentiation: cell adhesion, cell-cell signaling, and transcriptional regulation. 4. Bilaterally symmetrical animals, which represent the majority of animal lineages, possess a genetic toolkit for animal development, a select set of gene families involved in adhesion, cell communication, and differentiation. 5. Increasing evidence indicates that early animal history was an evolutionary radiation, suggesting that the exact relationships between early-branching phyla may be less important in understanding the origin and assembly of the genetic toolkit. 6. Five protist lineages are the closest relatives to animals, with the choanoﬂagellates, a clade of unicellular and colonial organisms, the most suitable for comparative purposes. 7. Examination of the DNA record of choanoﬂagellates and its comparison with that of early-branching, and bilaterian animals supports a model of gradual origins and assembly of the genetic toolkit, with different components originating and expanding at different time points prior to or soon after the origin of animals.

DISCLOSURE STATEMENT The author is not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS I am grateful to Nicole King and Sean B. Carroll for introducing me to choanoﬂagellates and the origins of multicellularity. Research in the Rokas lab is supported by the Searle Scholars Program

Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org and Vanderbilt University. Access provided by Houston Baptist University on 02/03/20. For personal use only.

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Annual Review of Genetics Contents Volume 42, 2008

Mid-Century Controversies in Population Genetics James F. Crow pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp1 Joshua Lederberg: The Stanford Years (1958–1978) Leonore Herzenberg, Thomas Rindﬂeisch, and Leonard Herzenberg ppppppppppppppppppppppp19 How Saccharomyces Responds to Nutrients Shadia Zaman, Soyeon Im Lippman, Xin Zhao, and James R. Broach pppppppppppppppppppp27 Diatoms—From Cell Wall Biogenesis to Nanotechnology Nils Kroeger and Nicole Poulsen pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp83 Myxococcus—From Single-Cell Polarity to Complex Multicellular Patterns Dale Kaiser pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp109 The Future of QTL Mapping to Diagnose Disease in Mice in the Age of Whole-Genome Association Studies Kent W. Hunter and Nigel P.S. Crawford ppppppppppppppppppppppppppppppppppppppppppppppppp131 Host Restriction Factors Blocking Retroviral Replication Daniel Wolf and Stephen P. Goff ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp143 Genomics and Evolution of Heritable Bacterial Symbionts Nancy A. Moran, John P. McCutcheon, and Atsushi Nakabachi ppppppppppppppppppppppppp165 Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org Access provided by Houston Baptist University on 02/03/20. For personal use only. Rhomboid Proteases and Their Biological Functions Matthew Freeman ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp191 The Organization of the Bacterial Genome Eduardo P.C. Rocha ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp211 The Origins of Multicellularity and the Early History of the Genetic Toolkit for Animal Development Antonis Rokas pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp235 Individuality in Bacteria Carla J. Davidson and Michael G. Surette pppppppppppppppppppppppppppppppppppppppppppppppp253

vii AR361-FM ARI 3 October 2008 13:37

Transposon Tn5 William S. Reznikoff pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp269 Selection on Codon Bias Ruth Hershberg and Dmitri A. Petrov ppppppppppppppppppppppppppppppppppppppppppppppppppppp287 How Shelterin Protects Mammalian Telomeres Wilhelm Palm and Titia de Lange ppppppppppppppppppppppppppppppppppppppppppppppppppppppppp301 Design Features of a Mitotic Spindle: Balancing Tension and Compression at a Single Microtubule Kinetochore Interface in Budding Yeast David C. Bouck, Ajit P. Joglekar, and Kerry S. Bloom pppppppppppppppppppppppppppppppppppp335 Genetics of Sleep Rozi Andretic, Paul Franken, and Mehdi Tafti pppppppppppppppppppppppppppppppppppppppppppp361 Determination of the Cleavage Plane in Early C. elegans Embryos Matilde Galli and Sander van den Heuvel ppppppppppppppppppppppppppppppppppppppppppppppppp389 Molecular Determinants of a Symbiotic Chronic Infection Kattherine E. Gibson, Hajime Kobayashi, and Graham C. Walker pppppppppppppppppppppp413 Evolutionary Genetics of Genome Merger and Doubling in Plants Jeff J. Doyle, Lex E. Flagel, Andrew H. Paterson, Ryan A. Rapp, Douglas E. Soltis, Pamela S. Soltis, and Jonathan F. Wendel ppppppppppppppppppppppppppppppppppppppppppppppppp443 The Dynamics of Photosynthesis Stephan Eberhard, Giovanni Finazzi, and Francis-Andr´e Wollman pppppppppppppppppppp463 Planar Cell Polarity Signaling: From Fly Development to Human Disease Matias Simons and Marek Mlodzik pppppppppppppppppppppppppppppppppppppppppppppppppppppppp517 Quorum Sensing in Staphylococci Richard P. Novick and Edward Geisinger pppppppppppppppppppppppppppppppppppppppppppppppppp541 Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org

Access provided by Houston Baptist University on 02/03/20. For personal use only. Weird Animal Genomes and the Evolution of Vertebrate Sex and Sex Chromosomes Jennifer A. Marshall Graves ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp565 The Take and Give Between Retrotransposable Elements and Their Hosts Arthur Beauregard, M. Joan Curcio, and Marlene Belfort ppppppppppppppppppppppppppppppp587 Genomic Insights into Marine Microalgae Micaela S. Parker, Thomas Mock, and E. Virginia Armbrust ppppppppppppppppppppppppppp619 The Bacteriophage DNA Packaging Motor Venigalla B. Rao and Michael Feiss ppppppppppppppppppppppppppppppppppppppppppppppppppppppppp647

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The Genetic and Cell Biology of Wolbachia-Host Interactions Laura R. Serbus, Catharina Casper-Lindley, Frédéric Landmann, and William Sullivan ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp683 Effects of Retroviruses on Host Genome Function Patric Jern and John M. Coffin pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp709 X Chromosome Dosage Compensation: How Mammals Keep the Balance Bernhard Payer and Jeannie T. Lee pppppppppppppppppppppppppppppppppppppppppppppppppppppppp733

Errata

An online log of corrections to Annual Review of Genetics articles may be found at http:// genet.annualreviews.org/errata.shtml Annu. Rev. Genet. 2008.42:235-251. Downloaded from www.annualreviews.org Access provided by Houston Baptist University on 02/03/20. For personal use only.

Contents ix